Mobility of four structural regions drives isoform-specific properties of photoenzyme LPOR in plants

Light-dependent protochlorophyllide oxidoreductase (LPOR) is a photocatalytic enzyme in the chlorophyll biosynthetic pathway that underwent duplications in angiosperms, resulting in the emergence of multiple isoforms across various plant species. The physiological roles of these LPOR homologs remained unclear, so we selected six plant species with different number of isoforms of the enzyme, having diverse phylogenetic backgrounds, and characterized their properties in vitro. Our findings revealed that these isoforms vary in their affinity for the reaction product, chlorophyllide (Chlide), as well as for NADPH under lipid-free conditions and in reaction mixtures supplemented with plant lipids. Additionally, we observed differences in their oligomerization behavior. Our experimental approach generated a dataset comprising several hundred pairs of spectra, recorded before and after reaction-triggering illumination. This data was used to analyze the correlation between fluorescence emission maxima before and after photoconversion. The analysis showed that some isoforms rapidly release Chlide after the reaction, while others retain the pigment in the binding pocket, especially at high NADPH concentrations. These results suggest that LPOR isoforms differ in their rates of Chlide release and complex disassembly, potentially influencing the overall rate of the chlorophyll biosynthetic pathway, even in mature leaves. We further analyzed the flexibility of these isoforms using AlphaFold2 predictions, identifying four regions of the enzyme that are particularly mobile. Two of these regions are involved in pigment binding, while the other two play a role in oligomerization. Based on these findings, we propose a model of conformational changes that drive the formation of LPOR oligomers.